Technetium-99M labeled peptides for thrombus imaging

ABSTRACT

This invention relates to radiolabeled peptides and methods for producing such peptides. Specifically, the invention relates to specific binding peptides, methods and kits for making such peptides, and methods for using such peptides labeled with technetium-99m via a radiolabel-binding moiety covalently linked to the peptide to image thrombus sites in a mammalian body.

This application is a continuation of application Ser. No. 07/886,752,filed May 21, 1992 abandoned, which is a continuation of Ser. No.07/653,012 abandoned, filed Feb. 8, 1991.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic reagents and peptides, andmethods for producing labeled radiodiagnostic agents. Specifically, theinvention relates to peptides that can be labeled with technetium-99m(Tc-99m), methods and kits for making and radiolabeling such peptides,and methods for using such peptides to image sites of thrombus formationin a mammalian body.

2. Description of the Prior Art

Thrombosis and thromboembolism, in particular deep vein thrombosis (DVT)and pulmonary embolism (PE), are common clinical conditions that areassociated with significant morbidity and mortality. It has beenestimated that in the U.S. approximately 5 million patients experienceone or more episodes of DVT per year and that over 500,000 cases ofpulmonary embolism occur, resulting in 100,000 deaths (J. Seabold,Society of Nuclear Medicine Annual Meeting 1990). It has also beenestimated that over 90% of all pulmonary emboli arise from DVT in thelower extremities. Anticoagulant therapy can effectively treat theseconditions if applied early enough. However, such treatment isassociated with risks (e.g. internal bleeding) that prevent unnecessaryprophylactic application. More advanced techniques of thrombolyticintervention (such as the administration of recombinant tissueplasminogen activator or streptokinase) can be used in acute cases, butthese techniques carry even greater risk. Moreover, effective clinicalapplication of these techniques requires that the site of the offendingthrombus be identified so as to monitor the effect of treatment.

For these reasons, a rapid means of localizing thrombi in vivo, mostpreferably using non-invasive methods, is highly desirable. Methodscurrently utilized for the identification of thrombolytic sites arecontrast venography and compression B-mode ultrasound; the choice ofwhich technique is used depends on the expected location of thethrombus. However the former technique is invasive and both techniquesare uncomfortable for the patient. In addition, these methods are inmany cases either unsuitable or yield inaccurate results.

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

In radioimaging, the radiolabel is a gamma-radiation emittingradionuclide and the radiotracer is located using a gamma-radiationdetecting camera (this process is often referred to as gammascintigraphy). The imaged site is detectable because the radiotracer ischosen either to localize at a pathological site (termed positivecontrast) or, alternatively, the radiotracer is chosen specifically notto localize at such pathological sites (termed negative contrast).

A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a radionuclide thatemits gamma energy in the 100 to 200 keV range is preferred. To minimizethe absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow.To allow for examinations to be performed on any day and at any time ofthe day, it is advantageous to have a source of the radionuclide alwaysavailable at the clinical site.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹⁶⁹ Yb or ¹⁸⁶Re. Tc-99m is a preferred radionuclide because it emits gamma radiationat 140 keV, it has a physical half-life of 6 hours, and it is readilyavailable on-site using a molybdenum-99/technetium-99m generator.

A gamma-emitting radiotracer that binds specifically to a component of athrombus in preference to other tissue when administered in vivo canprovide an external scintigraphic image which defines the location ofthe thrombus-bound radiotracer and hence the thrombus. There are severalpotential radiotracer targets in thrombi. Thrombi are constructs ofblood cells, largely platelets, enmeshed in cross-linked fibrin. Venousthrombi are fibrin-rich, whereas arterial thrombi are platelet-rich.Fibrin and platelets are thus obvious targets for designingradiopharmaceuticals for imaging thrombi, each having multiple possibletarget sites.

Activated platelets and fibrin have been used as targets in radioimagingthrombi because neither are normally found in circulating blood;circulating blood contains unactivated platelets and fibrinogen, afibrin precursor. Thrombus formation involves the proteolytic conversionof fibrinogen to fibrin and the physiological conversion of unactivatedplatelets to an activated state. Since little fibrin circulates in theblood stream (in contrast to its precursor, fibrinogen) and since mostcirculating platelets are unactivated, fibrin and activated plateletsare excellent and specific targets for imaging thrombi because they willnot be found to any substantial extent anywhere in vivo other than in athrombus.

The use of radiolabeled fibrinogen and radiolabeled platelets forradioimaging has a number of disadvantages, however. Blood andbackground tissue clearance of radiolabeled fibrinogen and platelets areslow, which necessitates a long delay between injection and imaging.Also, as thrombi age radiolabeled platelets become less efficientimaging agents, although fibrin and platelets already in an existingthrombus remain targets even in aged thrombi.

Attempts to provide radiotracers for imaging thrombi are known in theprior art. These include autologous platelets, labeled with either ¹¹¹In or ^(99m) Tc (Tc-99m), and ¹²³ I- and ¹²⁵ I-labeled fibrinogen (thelatter detected with a gamma scintillation probe as opposed to a gammacamera). In addition, other thrombus-associated components of thecoagulation system, such as enzymes (e.g. thrombin), proenzymes andother factors may be useful as thrombus-associated targets forradiotracers. Additional radiolabeled compounds used to label thrombiinclude plasmin, plasminogen activators, heparin, fibronectin, fibrinFragment E₁ and anti-fibrin and anti-platelet monoclonal antibodies seeKnight, 1990, Sem. Nucl. Med. 20: 52-67 for review!.

Of the methods of radiolabeling thrombi known in the prior art, themethods that have shown the most promise are radiolabeled platelets,radiolabeled antibodies and radiolabeled fibrin Fragment E₁. All ofthese have serious drawbacks with regard to their routine use.

The use of radiolabeled autologous platelets to image thrombi requiresthat autologous blood be drawn, the platelets then separated andradiolabeled under sterile conditions in addition, radiolabeling must beperformed so as to avoid activating the platelets!, and the radiolabeledplatelets then be readministered to the patient. Such radiolabeledplatelets have a long circulating time, resulting in poor target tonon-target ratios at early times, thereby requiring that radioimaging beperformed only after a delay of 24 to 72 hours. Moreover, aged thrombiare poorly visualized since such thrombi do not efficiently incorporatefresh platelets.

Radiolabeled antifibrin and antiplatelet monoclonal antibodies have alsobeen used in the prior art (typically to image DVT). The disadvantage tousing such reagents is that antibodies (and even antibody fragments)have slow blood and general tissue clearance characteristics and requirea delay of at least several hours for optimum imaging. In addition,immunological reagents have the capacity to induce an immune response inthe patient. Further, such reagents must be prepared from mammalian celllines (hybridomas) and thus carry the risk of contamination byinfectious human viruses.

Methods of using large radiolabeled peptides for imaging thrombi havebeen described in the prior art. For example, Fragment E₁ is a peptidederived from coagulated, cross-linked fibrin. It has been labeled with¹²³ I and Tc-99m to provide high quality images in humans.

Olexa et al., 1982, European Patent Application No. 823017009 disclose apharmaceutically acceptable radiolabeled peptide selected from FragmentE₁ isolated from cross-linked fibrin, Fragment E₂ isolated fromcross-linked fibrin, and peptides having an amino acid sequenceintermediate between Fragments E₁ and E₂. Unfortunately, this proteinfragment must be laboriously prepared from human fibrinogen, making itunsuitable for routine manufacture.

Hadley et al., 1988, PCT/US88/03318 disclose a method for detecting afibrin-platelet clot in vivo comprising the steps of (a) administeringto a patient a labeled attenuated thrombolytic protein, wherein thelabel is selectively attached to a portion of the thrombolytic proteinother than the fibrin binding domain; and (b) detecting the pattern ofdistribution of the labeled thrombolytic protein in the patient.

Sobel, 1989, PCT/US89/02656 discloses a method to locate the position ofone or more thrombi in an animal using radiolabeled, enzymaticallyinactive tissue plasminogen activator.

Peptides having the ability to bind to thrombi are known in the priorart. Ruoslahti & Pierschbacher, U.S. Pat. No. 4,578,079 describepeptides of sequence X-Arg-Gly-Asp-R-Y, wherein X and Y are either H oran amino acid, and R is Thr or Cys, the peptides capable of binding tothrombi in vivo.

Ruoslahti & Pierschbacher, U.S. Pat. No. 4,792,525 describe peptides ofsequence Arg-Gly-Asp-X, wherein X is Ser, Thr or Cys, the peptidescapable of binding to thrombi in vivo.

Hawiger et al., 1989, PCT/US89/01742 relates to peptides comprisingsequences for two binding sites of a protein.

Barker et al., 1991, PCT/US90/03788 disclose cyclic peptides forinhibiting platelet aggregation.

Radiolabeled peptides useful for radioimaging thrombi have been reportedin the prior art.

Ranby et al., 1988, PCT/US88/02276 disclose a method for detectingfibrin deposits in an animal comprising covalently binding aradiolabeled compound to fibrin.

Stuttle, 1990, PCT/GB90/00933 discloses radioactively labeled peptidescontaining from 3 to 10 amino acids comprising the sequencearginine-glycine-aspartic acid (RGD), capable of binding to an RGDbinding site in vivo.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

Maraganore et al., 1991, PCT/US90/04642 disclose a radiolabeled thrombusinhibitor comprising (a) a inhibitor moiety; (b) a linker moiety; and(c) and an "anion binding exosite (ABE)" binding site moiety.

The use of chelating agents for radiolabeling polypeptides, and methodsfor labeling peptides and polypeptides with Tc-99m are known in theprior art and are disclosed in co-pending U.S. patent applications Ser.Nos. 08/263,758 08/266,178 and U.S. Pat. No. 5,443,815 which are herebyincorporated by reference.

There remains a need for a small (to enhance blood and background tissueclearance), synthetic (to make routine manufacture practicable and toease regulatory acceptance) molecules radiolabeled with Tc-99m for usein imaging thrombi in vivo. Small synthetic peptides radiolabeled withTc-99m that bind specifically to components of thrombi fulfill this needand are provided by this invention.

SUMMARY OF THE INVENTION

The present invention provides scintigraphic imaging agents that areradioactively-labeled peptide reagents. Specifically, the inventionprovides peptide reagents for preparing thrombus imaging agents that aretechnetium-99m (Tc-99m) labeled. Thrombus imaging agents of theinvention are comprised of a specific binding peptide that specificallybinds to a thrombus in vivo that is covalently linked to a Tc-99mbinding moiety and labeled with Tc-99m.

In a first aspect of the present invention, the invention providespeptide reagents capable of being Tc-99m labeled for imaging thrombussites within a mammalian body, comprising a specific binding peptidehaving an amino acid sequence of 4-100 amino acids covalently linked toa Tc-99m binding moiety of formula:

    C(pgp).sup.s -(aa)-C(pgp).sup.s

wherein C(pgp)^(s) is a protected cysteine and (aa) is an amino acid. Ina preferred embodiment, the amino acid is glycine. In a preferredembodiment, the peptide is comprised between 4 and 30 amino acids.

In a second embodiment, the invention provides peptide reagents capableof being Tc-99m labeled for imaging thrombus sites within a mammalianbody, comprising a specific binding peptide having an amino acidsequence of 4-100 amino acids covalently linked to a Tc-99m bindingmoiety of formula:

    A--CZ(B)-- C(RR')!.sub.n --X

wherein

A is H, HOOC, H₂ NOC, or --NHOC;

B is SH or NHR";

X is H, methyl, SH or NHR";

Z is H or methyl;

R and R' are independently H or lower alkyl;

R" is H, lower alkyl or --C═O;

n is 0, 1 or 2;

and where B is NHR", X is SH, Z is H and n is 1 or 2; where X is NHR", Bis SH,Z is H and n is 1 or 2; where B is H, A is HOOC, H₂ NOC, or--NHOC, X is SH, Z is H and n is 0 or 1; where Z is methyl, X is methyl,A is HOOC, H₂ NOC, or --NHOC, B is SH and n is 0; and wherein the thiolmoiety is in the reduced form. In a preferred embodiment, the peptide iscomprised between 4 and 30 amino acids.

In another embodiment, the invention provides peptide reagents capableof being labeled with Tc-99m for imaging thrombus sites within amammalian body, comprising a specific binding peptide having an aminoacid sequence of 4-100 amino acids covalently linked to a Tc-99m bindingmoiety of formula: ##STR1## for purposes of this invention,radiolabel-binding moieties having this structure will be referred to aspicolinic acid (Pic)-based moieties!or ##STR2## wherein X is H or aprotecting group; (amino acid) is any amino acid and theradiolabel-binding moiety is covalently linked to the peptide. Forpurposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolylamine (Pica)-based moieties. Ina preferred embodiment, the amino acid is glycine and X is anacetamidomethyl protecting group. In additional preferred embodiments,the peptide is comprised between 4 and 30 amino acids.

Yet another embodiment of the invention provides peptide reagentscapable of being labeled with Tc-99m for imaging thrombus sites within amammalian body, comprising a specific binding peptide having an aminoacid sequence of 4-100 amino acids covalently linked to a Tc-99m bindingmoiety that is a bisamino bisthiol Tc-99m binding moiety. The bisaminobisthiol Tc-99m binding moiety in this embodiment of the invention has aformula selected from the group consisting of: ##STR3## wherein each Rcan be independently H, CH₃ or C₂ H₅ ; each (pgp)^(s) can beindependently a thiol protecting group or H; m, n and p areindependently 2 or 3; A is linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; and X ispeptide; and ##STR4## wherein each R is independently H, CH₃ or C₂ H₅ ;m, n and p are independently 2 or 3; A is linear or cyclic lower alkyl,aryl, heterocyclyl, combinations or substituted derivatives thereof; Vis H or CO-peptide; R' is H or peptide; provided that when V is H, R' ispeptide and when R' is H, V is peptide. For purposes of this invention,radiolabel-binding moieties having these structures will be referred toas "BAT" moieties. In a preferred embodiment, the peptide is comprisedbetween 4 and 30 amino acids.

The invention also provides thrombus imaging agents for imaging athrombus within a mammalian body comprising a specific binding peptidehaving an amino acid sequence of 4 to 100 amino acids and atechnetium-99m binding moiety covalently linked to the specific bindingpeptide, wherein the peptide is selected from the group consisting oflinear and cyclic peptides that are ligands for a GPIIa/IIIb receptorand do not comprise the amino acid sequence(arginine-glycine-aspartate), peptides that are ligands for apolymerization site of fibrin, and cyclic peptides comprising the aminoacid sequence (arginine-glycine-aspartate). In a preferred embodiment,the amino acid sequence of peptides that are ligands for apolymerization site of fibrin comprise multiple copies of the sequence(glycyl-prolyl-arginyl-prolyl).

The invention also comprises scintigraphic imaging agents that arecomplexes of the peptide reagents of the invention with Tc-99m andmethods for radiolabeling the peptide reagents of the invention withTc-99m. Radiolabeled complexes provided by the invention are formed byreacting the peptide reagents of the invention with Tc-99m in thepresence of a reducing agent. Preferred reducing agents include but arenot limited to dithionite ion, stannous ion and ferrous ion. Complexesof the invention are also formed by labeling the peptide reagents of theinvention with Tc-99m by ligand exchange of a prereduced Tc-99m complexas provided herein.

The invention also provides kits for preparing scintigraphic imagingagents that are the peptide reagents of the invention radiolabeled withTc-99m. Kits for labeling the peptide reagents of the invention withTc-99m are comprised of a sealed vial containing a predeterminedquantity of a peptide reagent of the invention and a sufficient amountof reducing agent to label the peptide with Tc-99m.

This invention provides methods for preparing peptide reagents of theinvention by chemical synthesis in vitro. In a preferred embodiment,peptides are synthesized by solid phase peptide synthesis.

This invention provides methods for using scintigraphic imaging agentsthat are Tc-99m labeled peptide reagents for imaging a thrombus within amammalian body by obtaining in vivo gamma scintigraphic images. Thesemethods comprise administering an effective diagnostic amount of Tc-99mlabeled peptide reagents of the invention and detecting the gammaradiation emitted by the Tc-99m label localized at the thrombus sitewithin the mammalian body.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides peptide reagents for preparingradiolabeled thrombus imaging agents for imaging a thrombus within amammalian body comprising an amino acid sequence covalently linked to atechnetium-99m binding moiety. For purposes of the invention, the termthrombus imaging reagent will refer to embodiments of the inventioncomprising a specific binding peptide covalently linked to a Tc-99mbinding moiety and labeled with Tc-99m.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) -Tc generator. Other radionuclides knownin the prior art have effective half-lives which are much longer (forexample, ¹¹¹ In, which has a half-life of 67.4 h) or are toxic (orexample, ¹²⁵ I).

In the Tc-99m binding moieties and peptides covalently linked to suchmoieties that contain a thiol covalently linked to a thiol protectinggroups (pgp)^(s) ! provided by the invention, the thiol-protectinggroups may be the same or different and may be but are not limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH--(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C--(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ --(4-methoxyphenyl);

--CH--(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃ -9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group acetamidomethylgroup.

Each specific-binding peptide provided by the invention is comprised ofa sequence of amino acids. The term amino acid as used in this inventionis intended to include all L-and D- amino acids, naturally occurring andotherwise. Specific-binding peptides provided by the invention includebut are not limited to peptides having the following sequences: ##STR5##

Specific-binding peptides of the present invention can be chemicallysynthesized in vitro. Peptides of the present invention can generallyadvantageously be prepared on an amino acid synthesizer. The peptides ofthis invention can be synthesized wherein the radiolabel-binding moietyis covalently linked to the peptide during chemical synthesis in vitro,using techniques well known to those with skill in the art. Suchpeptides covalently-linked to the radiolabel-binding moiety duringsynthesis are advantageous because specific sites of covalent linkagecan be determined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodimentscomprising picolinic acid (Pic-); e.g., Pic-Gly-Cys(protecting group)-!,the radiolabel-binding moiety can be synthesized as the last (i.e.,amino-terminal) residue in the synthesis. In addition, the picolinicacid-containing radiolabel-binding moiety may be covalently linked tothe ε-amino group of lysine to give, for example, αN(Fmoc)-Lys-εNPic-Gly-Cys(protecting group)!, which may be incorporated at anyposition in the peptide chain. This sequence is particularlyadvantageous as it affords an easy mode of incorporation into the targetbinding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety-Cys(protecting group)-Gly-Pica! can be prepared during peptidesynthesis by including the sequence -Cys(protecting group)-Gly-! at thecarboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

Examples of small synthetic peptides containing the Pic-Gly-Cys- and-Cys-Gly-Pica chelators are provided in the Examples hereinbelow. Thisinvention provides for the incorporation of these chelators intovirtually any peptide capable of specifically binding to a thrombus invivo, resulting in a radiolabeled peptide having Tc-99m held as neutralcomplex.

This invention also provides specific-binding small synthetic peptideswhich incorporate bisamine bisthiol (BAT) chelators which may be labeledwith Tc-99m. This invention provides for the incorporation of thesechelators into virtually any peptide capable of specifically binding toa thrombus in vivo, resulting in a radiolabeled peptide having Tc-99mheld as neutral complex. An examples of a small synthetic peptidecontaining a BAT chelator as radiolabel-binding moiety is provided inthe Examples hereinbelow.

In forming a complex of radioactive technetium with the reagents of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the reagent in the presence of a reducingagent. Preferred reducing agents are dithionite, stannous and ferrousions; the most preferred reducing agent is stannous chloride. Means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of areagent of the invention to be labeled and a sufficient amount ofreducing agent to label the reagent with Tc-99m. Alternatively, thecomplex may be formed by reacting a reagent of this invention with apre-formed labile complex of technetium and another compound known as atransfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the Tc-99m pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. An appropriate amount of thepeptide reagent is introduced into a vial containing a reducing agent,such as stannous chloride, in an amount sufficient to label the peptidewith Tc-99m. An appropriate amount of a transfer ligand as described(such as tartrate, citrate, gluconate or mannitol, for example) can alsobe included. The kit may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, buffers, preservatives and thelike. The components of the kit may be in liquid, frozen or dry form. Ina preferred embodiment, kit components are provided in lyophilized form.

Radiolabeled thrombus imaging reagents according to the presentinvention may be prepared by the addition of an appropriate amount ofTc-99m or Tc-99m complex into the vials and reaction under conditionsdescribed in Example 2 hereinbelow.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) to100 mCi per mL.

The thrombus imaging reagents provided by the present invention can beused for visualizing thrombi in a mammalian body when Tc-99m labeled. Inaccordance with this invention, the Tc-99m labeled peptide reagents areadministered in a single unit injectable dose. The Tc-99m labeledpeptide reagents provided by the invention may be administeredintravenously in any conventional medium for intravenous injection suchas an aqueous saline medium, or in blood plasma medium. Generally, theunit dose to be administered has a radioactivity of about 0.01 mCi toabout 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injectedat unit dosage is from about 0.01 mL to about 10 mL. After intravenousadministration, imaging of the thrombus in vivo can take place in amatter of a few minutes. However, imaging can take place, if desired, inhours or even longer, after the radiolabeled peptide is injected into apatient. In most instances, a sufficient amount of the administered dosewill accumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethyl-polystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid, water, thioanisole, ethanedithiol,and triethylsilane, prepared in ratios of 100:5:5:2.5:2 for 1.5-3 h atroom temperature.

Where appropriate, N-terminal acetyl groups were introduced by treatingthe free N-terminal amino peptide bound to the resin with 20% v/v aceticanhydride in NMP (N-methylpyrrolidinone) for 30 min. Where appropriate,2-chloroacetyl and 2-bromoacetyl groups were introduced either by usingthe appropriate 2-halo-acetic acid as the last residue to be coupledduring SPPS or by treating the N-terminus free amino peptide bound tothe resin with either the 2-halo-aceticacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP of the2-halo-acetic anhydride/ diisopropylethylamine in NMP. Whereappropriate, HPLC-purified 2-haloacetylated peptides were cyclized bystirring an 0.1-1.0 mg/mL solution in phosphate or bicarbonate buffer(pH 8) containing 0.5-1.0 mM EDTA for 4-48 hours, followed byacidification with acetic acid, lyophilization and HPLC purification.Where appropriate, Cys-Cys disulfide bond cyclizations were performed bytreating the precursor cysteine-free thiol peptides at 0.1 mg/mL in pH 7buffer with aliquots of 0.006M K₃ Fe(CN)₆ until a stable yellow colorpersisted. The excess oxidant was reduced with excess cysteine, themixture was lyophilized and then purified by HPLC.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS).

EXAMPLE 2 A General Method for Radiolabeling with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL ofwater or 50 mM potassium phosphate buffer (pH=5, 6 or 7.4). Tc-99mgluceptate was prepared by reconstituting a Glucoscan vial (E. I. DuPontde Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15-30min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using theconditions described in the Footnotes in Table I. Radioactive componentswere detected by an in-line radiometric detector linked to anintegrating recorder. Tc-99m gluceptate and Tc-99m sodium pertechnetateelute between 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 2 using the method described herein.

    __________________________________________________________________________                            FABMS                                                                             Radiochemical                                                                        HPLC                                       Peptides                MH.sup.+                                                                          Yield(%)*                                                                            R.sub.T (min)**                            __________________________________________________________________________     ##STR6##               1057                                                                              97.sup.2                                                                             10.0, 10.4, 10.6.sup.2                      ##STR7##               1357                                                                              100.sup.4                                                                            15.9, 16.4.sup.2                            ##STR8##               1318                                                                              97.sup.4                                                                             15.9, 16.3.sup.2                            ##STR9##               1310                                                                              99.sup.2                                                                             11.8.sup.2                                  ##STR10##              1171                                                                              99.sup.2                                                                             13.5.sup.2                                  ##STR11##              1233                                                                              100.sup.4                                                                            17.1, 18.1.sup.2                            ##STR12##              1200                                                                              96.sup.4                                                                             15.8, 16.1.sup.2                            ##STR13##              1217                                                                              70.sup.2                                                                             6.6-13.7.sup.2                              ##STR14##              1327                                                                              98.sup.2                                                                             13.0-15.5.sup.2                            Ac.CNP.Apc.GDC          832 99.sup.1                                                                             12.9.sup.2                                 C.sub.Acm GC.sub.Acm GGRGDS                                                                           953 100.sup.2                                                                            8.6.sup.1                                  C.sub.Acm GC.sub.Acm GGRGDGGRGDS                                                                      1396                                                                              100.sup.1                                                                            12.6.sup.1                                 C.sub.Acm GC.sub.Acm GGRGDGGRGDGGRGDS                                                                 1838                                                                              100.sup.2                                                                            10.0, 10.8.sup.1                           C.sub.Acm GC.sub.Acm RRRRRRRRRGD                                                                      2100                                                                              100.sup.2                                                                            2.4.sup.3***                               GRGDVKC.sub.Acm GC.sub.Acm amide                                                                      1036                                                                              100.sup.2                                                                            15.7.sup.2                                 GRGDVC.sub.Acm GC.sub.Acm amide                                                                       907 100.sup.2                                                                            16.1.sup.2                                 GRGDVRGDFKC.sub.Acm GC.sub.Acm amide                                                                  1510                                                                              97.sup.2                                                                             16.2, 16.8.sup.2                           GRGDVRGDFC.sub.Acm GC.sub.Acm amide                                                                   1382                                                                              94.sup.2                                                                             16.4.sup.2                                 (GPRVVERHQSA).sub.2 K   2986                                                                              99.sup.4                                                                             16.0.sup.2                                 CRGDC                   553 100.sup.3                                                                            16.7.sup.2                                 GRGDGGC                 769 98.sup.1                                                                             13.0, 13.6, 14.7.sup.2                     maGGRGDF                739 98.sup.1                                                                             13.8-14.7.sup.2                            mmpGGGRGDF              767 100.sup.3                                                                            18.4, 19.3.sup.2                           GRGDGGGGC               735 100.sup.3                                                                            14.9, 15.1, 15.4.sup.3                     maRGD                   421 97.sup.3                                                                             16.1, 16.9, 17.7.sup.2                     maRGDF                  568 94.sup.3                                                                             18.1, 18.7.sup.2                           CKRARGDDMDDYC           1548                                                                              97.sup.3                                                                             16.7.sup.2                                  Pic.SC.sub.Acm SYNRGDSTC(S-maleimido)CH.sub.2 CH.sub.2!.sub.3 N.sup.a                                4489                                                                              99.sup.2                                                                             10.4, 11.2.sup.2                           C.sub.Acm GC.sub.Acm NDGDFEEIPEELQ                                                                    2103                                                                              100.sup.2                                                                            2.5.sup.1****                              C.sub.Acm GC.sub.Acm GGF.sub.D PRPGGGGNGDFEEIPEEYL                                                    2699                                                                              99.sup.2                                                                             14.5.sup.3                                 maGGGF.sub.D PRPGGGGNGDFEEIPEEYL                                                                      2426                                                                              95.sup.2                                                                             17.4.sup.2                                 C.sub.Acm GC.sub.Acm GGF.sub.D PRPGamide                                                              1092                                                                              100.sup.5                                                                            9.6.sup.2                                   GPRPC.sub.Acm GC.sub.Acm C(S-maleimido)CH.sub.2 CH.sub.2!.sub.3 N.sup.b                              3189                                                                              93.sup.2                                                                             10.0.sup.2                                  (GPRP).sub.2 K!.sub.2 KC.sub.Acm GC.sub.Acm amide                                                    2437                                                                              100.sup.4                                                                            16.3.sup.2                                  BAT!G.Apc.GDV.Apc.GDFKamide                                                                          1432                                                                              96.sup.3                                                                             17.5.sup.2                                 __________________________________________________________________________     *Superscripts refer to the following labeling conditions:                     .sup.1 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       7.4) and labeled at room temperature.                                         .sup.2 The peptide is dissolved on 50 mM potassium phosphate buffer (pH       7.4) and labeled at 100° C.                                            .sup.3 The peptide is dissolved in water and labeled at room temperature.     .sup.4 The peptide is dissolved in water and labeled at 100° C.        .sup.5 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       6.0) and labeled at 100° C.                                            .sup.6 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       5.0) and labeled at room temperature.                                         **HPLC methods (indicated by superscript after R.sub.T):                      general:                                                                      solvent A = 0.1% CF.sub.3 COOOH/H.sub.2 O                                     solvent B.sub.70 = 0.1% CF.sub.3 COOOH/70% CH.sub.3 CN/H.sub.2 O              solvent B.sub.90 = 0.1% CF.sub.3 COOOH/90% CH.sub.3 CN/H.sub.2 O              solvent flow rate = 1 mL/min                                                  Vydak column = Vydak 218TP54 RP18, 5μ × 220 mm × 4.6 mm        analytical column with guard column Brownlee column = Spheri5 RP18, 5μ     × 220 mm × 4.6 mm column                                          Method 1:  Brownlee column  100% A to 100% B.sub.70 in 10 min                 Method 2:  Vydak column  100% A to 100% B.sub.90 in 10 min                    Method 3:  Vydak column  100% A to 100% B.sub.70 in 10 min                    ***Confirmed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis      ****Confirmed by binding the peptide to an affinity column                    Singleletter abbreviations for amino acid can be found in G. Zubay,           Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p.33; Ac =      acetyl; Pic = picolinoyl(pyridine2-carbonyl); Acm = acetamidomethyl; Mob      4methoxybenzyl; Apc = L S-(3-aminopropyl)cysteine; F.sub.D =                  Dphenylalanine; Y.sub.D = Dtyrosine; Cpa = L(4-chlorophenyl)alanine; ma =     2mercaptoacetic acid; mmp = 2mercapto-2-methylpropionic acid; BAT =           HSC(CH.sub.3).sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2          CH.sub.2 CH.sub.2 CO)CH.sub.2 C(CH.sub.3).sub.2 SH                            .sup.a The structure of this compound is as follows:                          ##STR15##                                                                     .sup.b The structure of this compound is as follows:                          ##STR16##                                                                

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 33                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=thioether                                       /note= "The sidechain sulfur of the C-terminal                                cysteine residue is covalently linked as an ether                             to the group  CH2CO!, which group forms an amide                              bond with the N-terminal D-tyrosine residue"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TyrArgGlyAspCys                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=thioether                                       /note= "The sidechain sulfur atom of the cysteine                             residue is covalently linked to the group  CH2CO!,                            which group forms an amide bond with the N-terminal                           D- tyrosine"                                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TyrArgGlyAspCysTrpGlyGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=thioether                                       /note= "The sidechain sulfur atom of the cysteine                             residue is covalently linked to the group  CH2CO!                             to form a thioether, and the  CH2CO!group is                                  covalently linked to the N-terminal D-tyrosine to                             form an amide bond."                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       TyrArgGlyAspCysPheGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=thioether                                       /note= "The sidechain sulfur atom of the cysteine                             is covalently linked to the group  CH2CO!to form                              a thioether; the  CH2CO!group is further linked                               to the N- terminal D-tyrosine to form an amide bond."                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TyrArgGlyAspCysGlyGlyGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=thioether                                       /note= "The sidechain sulfur atom of the cysteine                             is covalently linked to the group  CH2CO!to form                              a thioether; said group is further covalently                                 linked to the N-terminal D-tyrosine to form an amide                          bond."                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TyrArgGlyAspCysGlyGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label=thioether                                       /note= "The sulfur in the first cysteine is                                   covalently linked to a  3-aminopropyl group; the                              sulfur of the second cysteine is linked to a                                   CH2CO!group to form a thioether, and said                                     CH2CO!group is further covvalently linked                                    to the N- terminal D-tyrosine to form an amide bond."                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TyrCysGlyAspCysGlyGlyGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..5                                                            (D) OTHER INFORMATION: /label=thioether                                       /note= "The sidechain sulfur atom of the cysteine                             is covalently linked to the group  CH2CO!to form                              a thioether; this group then forms an amide bond                              with the N- terminal D-tyrosine residue"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TyrLysGlyAspCysGlyGlyGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Disulfide-bond                                                  (B) LOCATION: 3..9                                                            (D) OTHER INFORMATION: /label=Apc                                             /note= "The sidechain sulfur of the cysteine                                  residue at position 6 is covalently linked to a                                3- aminopropyl!group and hence is blocked"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GlyGlyCysAsnProCysGlyAspCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GlyGlyArgGlyAspSer                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GlyGlyArgGlyAspGlyGlyArgGlyAspSer                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GlyGlyArgGlyAspGlyGlyArgGlyAspGlyGlyArgGlyAspSer                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      LysArgAlaArgGlyAspAspMetAspAspTyr                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgArgArgArgArgArgArgArgArgGlyAsp                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyArgGlyAspValLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GlyArgGlyAspVal                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlyArgGlyAspValArgGlyAspPheLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GlyArgGlyAspValArgGlyAspPhe                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GlyGlyGlyArgGlyAspPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /label=Apc                                             /note= "The sidechain sulfur of the cysteine                                  residue is covalently linked to a  3-aminopropyl!                             group forming a thioether"                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      AsnProCysGlyAsp                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GlyArgGlyAspGlyGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GlyGlyArgGlyAspPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GlyGlyGlyArgGlyAspPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GlyArgGlyAspGlyGlyGlyGly                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GlyArgGlyAspGlyGly                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GlyGlyArgGlyAspPhe                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      GlyGlyGlyArgGlyAspPhe                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 2                                                               (D) OTHER INFORMATION: /label=Apc                                             /note= "The sidechain sulfur atom is covalently                               linked to a  3-aminopropyl!group to form a                                    thioether"                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 6                                                               (D) OTHER INFORMATION: /label=Apc                                             /note= "The sidechain sulfur atom is covalently                               linked to a  3-aminopropyl!group to form a                                    thioether"                                                                    (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 10                                                              (D) OTHER INFORMATION: /label=Amide                                           /note= "The carboxyl terminus is modified to an                               amide"                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GlyCysGlyAspValCysGlyAspPheLys                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /label= S- maleimido                                   /note= "The sidechain sulfur atom is covalently                               linked to a  S-maleimido!group; 3 such peptides                               are covalently linked to a nitrogen atom to form a                            tertiary amine"                                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      SerTyrAsnArgGlyAspSerThrCys                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      AsnAspGlyAspPheGluGluIleProGluGluTyrLeuGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /label= D- Phe                                         /note= "This residue is the D stereoisomer"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GlyGlyPheProArgProGlyGlyGlyGlyAsnGlyAspPheGluGlu                              151015                                                                        IleProGluGluTyrLeu                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 4                                                               (D) OTHER INFORMATION: /label= D- Phe                                         /note= "This residue is the D stereoisomer"                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GlyGlyGlyPheProArgProGlyGlyGlyGlyAsnGlyAspPheGlu                              151015                                                                        GluIleProGluGluTyrLeu                                                         20                                                                            (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 3                                                               (D) OTHER INFORMATION: /label= D- Phe                                         /note= "This residue is the D stereoisomer"                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /label=Amide                                           /note= "The carboxyl terminus is modified to an                               amide"                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GlyGlyPheProArgProGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /label=Acm                                             /note= "This cysteine residue is blocked at the                               sidechain sulfur by covalent linkage to an                                    acetamido group"                                                              (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 7                                                               (D) OTHER INFORMATION: /label=Acm                                             /note= "This cysteine residue is blocked at the                               sidechain sulfur atom by covalent linkage to an                               acetamido group"                                                              (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 8                                                               (D) OTHER INFORMATION: /label= S- maleimido                                   /note= "The sidechain sulfur atom of this cysteine                            residue is covalently linked to an  S-maleimido!                              group; 3 such peptides are linked via this group                              to a nitrogen atom to form a tertiary amine"                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      GlyProArgProCysGlyCysCys                                                      15                                                                            __________________________________________________________________________

What is claimed is:
 1. A reagent for preparing a thrombus imaging agentfor imaging a thrombus within a mammalian body comprising a specificbinding peptide and a technetium-99m binding moiety covalently linked tothe specific binding peptide, wherein the peptide binds to a componentof a thrombus, the technetium-99m binding moiety having formula:

    C(pgp).sup.s -(aa)-C(pgp).sup.s

wherein C(pgp)^(s) is a cysteine having a protected thiol group and (aa)is an amino acid.
 2. The reagent of claim 1 wherein the specific bindingpeptide and C(pgp)^(s) -(aa)-C(pgp)^(s) are covalently linked throughfrom about one to about twenty amino acids.
 3. The reagent of claim 1wherein the protected cysteine has a protecting group of the formula

    --CH.sub.2 --NH--CO--R

wherein R is a lower alkyl having 1 to 6 carbon atoms, 2-,3-,4-pyridyl,phenyl, or phenyl substituted with lower alkyl, hydroxy, lower alkoxy,carboxy, or lower alkoxycarbonyl.
 4. The reagent of claim 1 whereinC(pgp)^(s) -(aa)-C(pgp)^(s) has the formula: ##STR17##
 5. The reagent ofclaim 1 having the formula: ##STR18##
 6. The reagent of claim 1 havingthe formula ##STR19##
 7. A kit for preparing a radiopharmaceuticalpreparation, said kit comprising a sealed vial containing apredetermined quantity of the reagent of claim 1 and a sufficient amountof reducing agent to label the reagent with technetium-99m.
 8. Thereagent according to claim 1 wherein the specific-binding peptide ischemically synthesized in vitro.
 9. The reagent according to claim 8wherein the specific-binding peptide is synthesized by solid phasepeptide synthesis.
 10. The reagent according to claim 8 wherein theradiolabel-binding moiety is covalently linked to the specific-bindingpeptide during in vitro chemical synthesis.
 11. The reagent according toclaim 10 wherein the radiolabel-binding moiety is covalently linked tothe specific-binding peptide during solid phase peptide synthesis.
 12. Acomposition of matter comprising the reagent according to claim 1wherein the specific binding peptide is selected from the groupconsisting of peptides having the amino acid sequence: ##STR20##